Cell-autonomous effect of cardiomyocyte branched-chain amino acid catabolism in heart failure in mice

Figures

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  Fig. 1. Generation of cardiac specific BCAA metabolism defective mouse model using BCKDHA-cKO mice. a Schematic view of BCKDH complex in BCAA catabolic pathway and the experimental design. b Bckdha mRNA levels in the Control and the BCKDHA-cKO mouse hearts measured by real-time RT-PCR, n = 7–10 each group, ****P < 0.001. c Representative immunoblot data of total and phosphorylated E1α protein, and E2 protein in the BCKDHA-cKO and the Control mouse hearts. d Representative immunoblot data of E1α expression in liver and heart tissues from the Control and the BCKDHA-cKO mice. e Real-time PCR analysis of BCKDHA normalized to GAPDH in different tissues as indicated. n = 3 each group, *P < 0.05.

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  Fig. 2. Impaired cardiac function following BCAA catabolic defect in heart. a Body weight of the male and the female Control and BCKDHA-cKO mice at baseline (Day 0) and one week post tamoxifen injection (Day 12). b Representative echo M-mode images for the Control and the BCKDHA-cKO mice at baseline and post tamoxifen mediated gene inactivation. c–h Echocardiography parameters, including (c) ejection fraction (EF%); (d) fractional shortening (FS%); (e) left ventricle end-diastolic volume (LV Vol;d); (f) left ventricle end-systolic volume (LV Vol;s); (g) left ventricle end-diastolic internal diameter (LVID;d) and (h) left ventricle end-systolic internal diameter (LVID;s) in the Control and the BCKDHA-cKO male and female animals at baseline (Day 0) and one week post-tamoxifen induced gene inactivation (Day 12). **P < 0.01; ****P < 0.001.

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  Fig. 3. Pathological remodeling following BCAA catabolic defect in heart. a Representative H&E staining of the Control and the BCKDHA-cKO male and female mice cardiac section. Magnification: 10×. b Cardiomyocytes cross-section area in the Control and the BCKDHA-cKO hearts, ***P < 0.005. c, d Heart weight vs. tibial length (HW/TL), Left ventricle weight vs. tibial length (LV/TL) in the Control and the BCKDHA-cKO animals. e–h mRNA levels of ANF (e, f) and BNP relative to ACTB (g, h) in the Control and the BCKDHA-cKO male and female mouse hearts (e, g) as well as their quantitative correlation with cardiac function in each cohort (f, h).

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  Fig. 4. Impaired cardiac BCAA metabolism led to changes of systemic BCAA/BCKA. a BCAA, pyruvate and BCKA concentrations in the Control and the BCKDHA-cKO mouse cardiac tissues collected at one week post tamoxifen administration. b BCAA, pyruvate and BCKA concentrations in the Control and BCKDHA-cKO mouse serum samples collected at one week post tamoxifen administration. *P < 0.05, **P < 0.01, ****P < 0.001.

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  Fig. 5. Generation of cardiac specific BCAA metabolism active mouse via BCKDK-cKO mouse line. a Schematic view of the BCKDK-cKO mouse line generation and experimental design. b Real-time PCR analysis of BCKDK mRNA expression level in the Control and the BCDK-cKO male and female mouse left ventricle tissues. n = 4–5 each group, ****P < 0.001. c Western Blot analysis of BCKDK, E1α, phosphor-E1α (S293) and E2 expression level in the Control and the BCKDK-cKO mouse left ventricle tissues, GAPDH was used as control.

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  Fig. 6. Inactivation of BCKDK in heart does not prevent heart failure post pressure overload. a–f Echocardiography parameters including Ejection Fraction (a), Fraction Shortening (b), LVID;s (c), LVID;d (d), LV Vol;s (e) and LV Vol; d(f) in the Control and the BCKDK-cKO male and female mice, at baseline and 6 weeks post pressure overload injury. g Representative H&E staining for the Control and the BCKDK-cKO female and male mouse hearts post pressure overload surgery. Magnification: 10×. h Heart weight/tibia length ratio in the Control and the BCKDK-cKO male and female mice 6 weeks post TAC. i Left ventricle weight/tibia length ratio in the Control and the BCKDK-cKO male and female mice 6 weeks post TAC. j–k: Survival curve for the Control and the BCKDK-cKO female (j) and male (k) mice post TAC at indicated time points. **P < 0.01.

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  Fig. 7. Transcriptome changes in BCKDHA-cKO and BCKDK-cKO mouse hearts. a Heatmap of selected genes detected in the Control vs the BCKDHA-cKO hearts from RNA-seq dataset ranked based on their expression levels in the BCKDHA-cKO hearts. b Integrated biological pathway and Gene-Ontology analysis for the top 500 differentially regulated genes ranked by significance in P values were determined by ClueGO in the Control and the BCKDHA-cKO hearts. c Heatmap of selected genes detected in the Control vs the BCKDK-cKO hearts from RNA-seq dataset ranked based on their expression levels in BCKDK-cKO hearts. d Integrated biological pathway and Gene-Ontology analysis for the top 500 differentially regulated genes ranked by significance in P values were determined by ClueGO in the Control and the BCKDK-cKO hearts. Colors represent related GO term families and only GO terms with q < 1E-20 are included.